The broad objective of the proposed research is to develop and exploit nuclear magnetic resonance (NMR) techniques for the study of protein structure and function in biological membranes. We have prepared seventeen different cytochrome c derivatives in each of which a single lysine group has been specifically modified with a fluorine containing reagent. All surface areas on cytochrome c have been labelled with these F-19 NMR probes. F-19 NMR methods will be used to accurately locate the binding sites on cytochrome c for cytochrome oxidase, cytochrome c1, and phospholipid membranes. We will investigate possible conformational changes at these binding sites due to electron transfer, and also attempt to measure distances between the F-19 labels on cytochrome c and paramagnetic metals in the oxidase and cytochrome c1. This information is very important for an understanding of what types of electron transfer mechanisms might be feasible. F-19 NMR methods will be used to study the interaction of the cytochrome c derivatives with intact mitochrondria. These studies should allow us to determine what components of the mitochondrial membrane cytochrome c is binding to under different physiological conditions. A central question is whether cytochrome c can bind to cytochrome oxidase and cytochrome reductase simultaneously, thus transporting electrons directly, or whether the binding sites are competitive and cytochrome c must undergo rotational and translational motion to transport an electron from cytochrome reductase to cytochrome oxidase. We will also carry out a detailed study of the cytochrome b 5-cytochrome c complex, which is the only such system in which both components have been characterized by x-ray crystallography.